Placental tissue protein PP4, proteins PP4-X, PAP III and p68, as well as lipocortins I and II, display homology in the amino acid sequences and belong to a family of proteins called lipocortins.
These proteins have antiinflammatory and anticoagulant effects. They have been detected in many organs and can be isolated from the latter.
Preparation from tissues, for example from human placenta, or of proteins prepared by gene manipulation, for example expressed in E. coli, such as rPP4 or rPP4-X, is associated with isolation, together with these proteins, of substances which are toxic for humans, such as bacterial lipopolysaccharides. Despite high purity (greater than 95% based on the protein content), the isolated proteins showed heavy contamination with toxic substances in toxicity tests such as the Limulus test or after administration of therapeutic doses (1 mg of protein/kg of body weight) to rabbits.
It was not possible to remove these evidently protein-associated contaminants either by chromatographic processes or filtration techniques such as sterile filtration or by the use of non-ionic detergents or of chelating reagents, alone or in combination.
Hence the object of the invention was to develop processes for removing toxins deriving from organs, tissues and cell cultures in proteins of the lipocortin family which have been isolated as well as prepared by gene manipulation, which do not impair the biological activity of the proteins and thus permit potential use as therapeutics for coagulation and/or inflammatory disorders.
It has been found, surprisingly, that toxic substances can be removed from these proteins by ion exchange chromatography in the presence of chelating reagents in combination with ionic detergents, without an adverse effect on the biological activity.